We have isolated full length or partial cDNA clones encoding four novel rat protein kinases related to the yeast protein kinase STE2O. In Saccharomyces, STE2O functions downstream of heterotrimeric G protein beta-gamma subunits but upstream of enzymes in a MAP kinase module involved in the pheromone response pathway. One of the kinases is highly similar to PAK1, recently purified and cloned by Lim's group; PAK1 is activated by the small GTP binding protein Rac1. The relationship of STE20 to the MAP kinase pathway and of PAK1 to small G proteins leads to the hypothesis that the four novel rat protein kinases may be involved in regulation of the mammalian MAP kinase cascade and may mediate some of the actions of the Rho/Rac family of small G proteins to control organization of the actin cytoskeleton and other biological events. To understand the function and regulation of these STE20-related protein kinases, we will immunolocalize the enzymes in cultured cells, examine their ability to influence MAP kinase activity, cell proliferation, differentiation, the actin cytoskeleton, and study how their activities are regulated. We will investigate the relationship between these kinase and small G proteins by determining how small G proteins affect kinase activity in vitro and by linking the actions of small G proteins that interact with the kinases to the effects of the kinases in intact cells. Finally, we will use the two- hybrid system and in vitro reconstitution to identify substrates and novel regulators of the STE20 like kinases.